scholarly journals Characterization of Saccharomyces cerevisiae CYP51 and a CYP51 fusion protein with NADPH cytochrome P-450 oxidoreductase expressed in Escherichia coli.

1997 ◽  
Vol 41 (4) ◽  
pp. 776-780 ◽  
Author(s):  
K Venkateswarlu ◽  
D E Kelly ◽  
S L Kelly
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Reductase Activity ◽  
Spectral Response ◽  
Cumene Hydroperoxide ◽  
Type Ii ◽  
Nadph Cytochrome ◽  
Binding Spectra ◽  
Cytochrome P 450 ◽  
Fus Protein

Saccharomyces cerevisiae CYP51, target of azole antifungal agents, and CYP51 fused with S. cerevisiae cytochrome P-450 oxidoreductase (FUS protein) were expressed in active forms in Escherichia coli by cloning into pET15b. The expression was monitored immunologically, catalytically, and by using reduced carbon monoxide difference and type II binding spectra. CYP51 and FUS enzymes were located in membranes and produced a Soret peak at 448 nm in the reduced CO difference spectrum. The cytochrome P-450 contents in the membrane fractions containing CYP51 and FUS proteins were 12.8 +/- 2.6 and 17.4 +/- 3.7 pmol/mg of protein, respectively. The NADPH cytochrome P-450 oxidoreductase (CPR) content was estimated to be 15.7 +/- 1.1 pmol/mg of protein in FUS membrane fractions. FUS protein catalyzed the demethylation of substrate at the 14alpha position, with a turnover number of 1.96 +/- 0.37 min(-1) in the presence of NADPH. No reductase activity was observed in membrane fractions containing CYP51, and therefore, CYP51 did not function catalytically in the presence of NADPH, but in the presence of an artificial electron donor, cumene hydroperoxide, activity was comparable to that of the FUS enzyme. Further support for a normal structure for the hemoproteins was obtained from type II binding spectra, in which the spectral response was saturated with an equimolar concentration of ketoconazole.

scholarly journals Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants

1989 ◽  
Vol 259 (3) ◽  
pp. 847-853 ◽  
Author(s):  
I Benveniste ◽  
A Lesot ◽  
M P Hasenfratz ◽  
F Durst
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Higher Plants ◽  
Polyclonal Antibodies ◽  
Reductase Activity ◽  
Jerusalem Artichoke ◽  
Cross Reactivity ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Cytochrome P 450

Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.

scholarly journals Presence of nadph-cytochrome P-450 reductase in rat liver golgi membranes: Evidence obtained by immunoadsorption method

1978 ◽  
Vol 79 (2) ◽  
pp. 590-597 ◽  
Author(s):  
A Ito ◽  
GE Palade
Keyword(s):  
Rat Liver ◽  
Reductase Activity ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Nadph Cytochrome ◽  
Golgi Membranes ◽  
Cyt C ◽  
Bona Fide ◽  
Liver Homogenates ◽  
Cytochrome P 450

Light Golgi fractions (GF(1+2)) prepared from rat liver homogenates by a modification of the Ehrenreich et al. procedure (J. Cell Biol. 59:45) had significant NADPH-cytochrome P(450) reductase (NADPH-cyt c reductase) activity if assayed immediately after their isolation. An antibody raised in rabbits against purified microsomal and Golgi fractions. To find out whether this activity is located in bona fide Golgi elements or in contaminating microsomal vesicles, we used the following 3-step immunoadsorption procedure: (a) antirabbit IgG (raised in goats) was conjugated to small (2-5 μm) polycrylamide (PA) beads; (b) rabbit anti NADPH-cyt c reductase was immunoadsorbed to the antibody-coated beads; and (c) GF(1+2) was reacted with the beads carrying the two successive layers of antibodies. The beads were then recovered by centrifugation, and were washed, fixed, embedded in agarose, and processed for transmission electromicroscopy. Antireductase- coated beads absorbed 60 percent of the NADPH-cyt c reductase (and comparable fractions of NADH-cyt c reductase and glucose-6-phosphatase) but only 20 percent of the galactosyltransferase activity of the input GF(1+2). Differential vesicle counts showed that approximately 72 percent of the immunoadsorbed vesicles were morphologically recognizable Golgi elements (vesicles with very low density lipoprotein [VLDL] clusters or Golgi cisternae); vesicles with single VLDL and smooth surfaced microsome-like vesicles were too few (approximately 25 percent) to account for the activity. It is concluded that NADPH-cytochrome P(450) reductase is a Golgi membrane enzyme of probably uneven distribution among the elements of the Golgi complex.

Temperature Influence on Basal Activity and Induction of Mixed Function Oxygenase Activity in Fundulus heteroclitus

1979 ◽  
Vol 36 (11) ◽  
pp. 1400-1405 ◽  
Author(s):  
John J. Stegeman
Keyword(s):  
Cytochrome C ◽  
Fundulus Heteroclitus ◽  
Reductase Activity ◽  
Control Fish ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Pyrene Hydroxylase ◽  
Mixed Function Oxygenase ◽  
Cytochrome P 450 ◽  
Nadph Cytochrome C Reductase

Treatment of Fundulus heteroclitus acclimated to 6.5 °C with benzo(a)pyrene did not elicit any change in the levels of hepatic microsomal NADH- or NADPH-cytochrome c reductase activity, nor in the levels of cytochrome P-450 or its catalytic activities. However, the same treatment offish at 16 5 °C resulted in a marked induction of benzo(a)pyrene hydroxylase and NADPH-cytochrome c reductase. Cytochrome P-450 content was also higher in the warm, treated fish and the Soret maximum of reduced, CO-treated microsomes was shifted to the violet. Levels of aminopyrine demethylase and NADH-cytochrome c reductase activities did not show a significant treatment effect. At neither temperature could treated and control fish be distinguished on the basis of in vitro inhibition of benzo(a)pyrene hydroxylase activity by 7,8-benzoflavone. Levels of NADPH-cytochrome c reductase and benzo(a)pyrene hydroxylase activities were greater in control Fundulus acclimated to 6.5 °C than to 16.5 °C, when normalized to microsomal protein, but not when based on body weight. The results indicate that habitat temperature alone may not affect the capacity for initial hydrocarbon metabolism in fish, but that it can strongly influence the induction of cytochrome P-450. Key words: temperature, cytochrome P-450, hydrocarbon metabolism, mixed-function oxygenase, Fundulus heteroclitus

scholarly journals The distribution of the components of mixed-function oxidase between the rough and the smooth endoplasmic reticulum of liver cells

1968 ◽  
Vol 110 (3) ◽  
pp. 407-412 ◽  
Author(s):  
J. L. Holtzman ◽  
T. E. Gram ◽  
P. L. Gigon ◽  
J. R. Gillette
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Reductase Activity ◽  
Mixed Function Oxidase ◽  
Protein Ratio ◽  
Nadph Cytochrome ◽  
Rate Limiting Step ◽  
Significant Difference ◽  
The Difference ◽  
Cytochrome P 450

Mixed-function oxidase activity, when measured by the N-demethylation of ethylmorphine or the hydroxylation of aniline, is significantly higher in the smooth hepatic endoplasmic reticulum than in the rough. In the rabbit the smooth membrane/rough membrane activity ratios are significantly greater than 1 whether the activities are expressed per g. of liver (ratio 5), per mg. of protein (ratio 3–5), per μg. of phospholipid phosphorus (ratio 2), per unit of cytochrome P-450 (ratio 1·7) or per unit of NADPH–cytochrome c reductase activity (ratio 2). On the other hand, if the activities are normalized to the NADPH–cytochrome P-450 reductase, there is no significant difference between the rough and smooth membranes. These results suggest that, in the rabbit, the rate-limiting step is the reduction of cytochrome P-450. In contrast, in the rat the difference in activities can be explained by differences in the concentration of cytochrome P-450.

Sign in / Sign up

Export Citation Format

Share Document